Members of the kinesin superfamily of mechanoenzymes move along microtubules in the course of carrying out their many critical functions in the cell. X-ray structures of both plus-end directed and minus-end directed kinesins are now available, as is the high resolution electron crystallographic structure of the tubulin protofilament. Despite much effort, there has been no success in preparing crystals of the track- motor complexes for crystallographic analyses. Combining the high resolution structures of the components with low resolution (20-30A) 3D maps obtained by cryo-electron microscopy is the only way to understand the detailed workings of the assemblies at various stages in the cycle of interaction. Studies will focus on three members of the kinesin superfamily: a conventional dimeric, plus-end directed motor (brain kinesin), a dimeric minus-end directed motor (ncd), and a monomeric plus-end directed motor (Unc104). Cryo-EM and image analysis of gold-cluster labeled proteins, coupled with difference mapping and modeling will be used to: - understand the near-atomic interactions of kinesins and tubulin, - visualize motions within the core motor domain, - visualize the movements of part of the neck region, - understand how the two heads of dimeric molecules are coordinated. These data will provide a detailed structural understanding of how three members of the kinesin superfamily produce directional movement. This work falls into the category of Basic Biomedical Research and has a basic and fundamental relevance for both the healthy and diseased states.
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